Isolation and characterization of tumor cells using shear stress measurements

ABSTRACT

Methods for isolating viable cancer cells from a sample that comprises a mixture of cancerous cells and normal (non-cancerous) cells are provided. In the methods, a fluid preparation comprising a mixture of cancerous and normal cells is repeatedly exposed to fluid shear stresses, whereby the repeated exposure to the fluid shear stresses preferentially imparts fluid shear stress-resistance to the cancerous cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/006,761, filed Oct. 15, 2013, which is a National Stage Entry under35 U.S.C. §371 of International patent application numberPCT/US2012/030034, filed Mar. 22, 2012, which claims the benefit of U.S.provisional patent application No. 61/466,983, filed on Mar. 24, 2011,the entire contents of which are all incorporated herein by reference.

REFERENCE TO GOVERNMENT RIGHTS

This invention was made with government support under RO1 CA130916awarded by the National Institutes of Health and under grant numberW81XWH-10-1-0313 awarded by the U.S. Army Medical Research and MaterialCommand. The government has certain rights in this invention.

BACKGROUND

Metastasis is the leading cause of mortality in patients with epithelialcancers. This complex process involves the detachment of cells from theprimary tumor, invasion into surrounding tissue, entrance to andsurvival within the bloodstream, extravasation, and, finally, survivaland proliferation at the secondary site. It is believed that metastasisis an inefficient process. Clinically, metastatic inefficiency can beappreciated by considering that many tumors continuously shed cancercells into the bloodstream on a daily basis, giving rise to a populationof circulating tumor cells (CTCs), yet only a small number of these goon to colonize distant sites in a process which may take decades.

Some groups have reported that survival within the bloodstream andsubsequent extravasation are completed efficiently by most tumor cellsand that the ability to survive and grow at secondary sites is whatdetermines the aggressiveness of a cell type [Luzzi, K. J., et al.,Multistep nature of metastatic inefficiency: dormancy of solitary cellsafter successful extravasation and limited survival of earlymicrometastases. Am J Pathol, 1998. 153(3): p. 865-73., Koop, S., etal., Fate of melanoma cells entering the microcirculation: over 80%survive and extravasate. Cancer Res, 1995. 55(12): p. 2520-3.,Podsypanina, K., et al., Seeding and propagation of untransformed mousemammary cells in the lung. Science, 2008. 321(5897): p. 1841-4., Tsuji,T., et al., Epithelial-mesenchymal transition induced by growthsuppressor p12CDK2-AP1 promotes tumor cell local invasion but suppressesdistant colony growth. Cancer Res, 2008. 68(24): p. 10377-86.]. In aneffort to study the fate of CTCs, other groups have conducted studies tomonitor the destination and viability of tumor cells injectedsystemically into mice. These authors concluded that the majority ofcirculating tumor cells are rapidly destroyed in the bloodstream byshear force [Fidler, I. J., Metastasis: quantitative analysis ofdistribution and fate of tumor embolilabeled with 125I-5-iodo-2′-deoxyuridine. J Natl Cancer Inst, 1970. 45(4): p. 773-82.,Fidler, I. J., Biological behavior of malignant melanoma cellscorrelated to their survival in vivo. Cancer Res, 1975. 35(1): p.218-24.] and/or by deformation following size restriction in themicrovasculature [Weiss, L., Deformation-driven, lethal damage to cancercells. Its contribution to metastatic inefficiency. Cell Biophys, 1991.18(2): p. 73-9., Weiss, L., et al., Lethal deformation of cancer cellsin the microcirculation: a potential rate regulator of hematogenousmetastasis. Int J Cancer, 1992. 50(1): p. 103-7.]. This led to thelongstanding assumption that cell death within the circulation is amajor contributor to metastatic inefficiency. Observations ofsignificant cell loss following injection into mice have been reportedby others as well [Kienast, Y., et al., Real-time imaging reveals thesingle steps of brain metastasis formation. Nat Med, 2010. 16(1): p.116-22., Al-Mehdi, A. B., et al., Intravascular origin of metastasisfrom the proliferation of endothelium-attached tumor cells: a new modelfor metastasis. Nat Med, 2000. 6(1): p. 100-2.].

During hematogenous dissemination, CTCs encounter a wide range of shearstresses (1-10⁵ dyn/s) [Schneider, S. W., et al., Shear-inducedunfolding triggers adhesion of von Willebrand factor fibers. Proc NatlAcad Sci U S A, 2007. 104(19): p. 7899-903., Reneman, R. S., T. Arts,and A. P. Hoeks, Wall shear stress—an important determinant ofendothelial cell function and structure—in the arterial system in vivo.Discrepancies with theory. J Vasc Res, 2006. 43(3): p. 251-69.]. Shearstress is a major component of the vascular microenvironment and hasimportant biological implications; for example, endothelial cells arefine-tuned to shear stress and variations in the magnitude or frequencyof shear forces have effects on the signaling, gene expression, andsurvival of these cells [Malek, A. M., S. L. Alper, and S. Izumo,Hemodynamic shear stress and its role in atherosclerosis. JAMA, 1999.282(21): p. 2035-42., Malek, A. and S. Izumo, Physiological fluid shearstress causes downregulation of endothelin-1 mRNA in bovine aorticendothelium. Am J Physiol, 1992. 263(2 Pt 1): p. C389-96.]. Shear stresshas also been shown to induce changes in the gene expression andadhesive properties of both leukocytes and cancer cells [Okuyama, M., etal., Fluid shear stress induces actin polymerization in humanneutrophils. J Cell Biochem, 1996. 63(4): p. 432-41., Avvisato, C. L.,et al., Mechanical force modulates global gene expression andbeta-catenin signaling in colon cancer cells. J Cell Sci, 2007. 120(Pt15): p. 2672-82., Stroka, K. M. and H. Aranda-Espinoza, A biophysicalview of the interplay between mechanical forces and signaling pathwaysduring transendothelial cell migration. FEBS J, 2010. 277(5): p.1145-58.]. Epithelial cells, from which carcinomas are derived, residein environments with much lower shear stress than found in thebloodstream [Althaus, M., et al., Mechano-sensitivity of epithelialsodium channels (ENaCs): laminar shear stress increases ion channel openprobability. The FASEB journal: official publication of the Federationof American Societies for Experimental Biology, 2007. 21(10): p.2389-99.]. It is thus reasonable to believe such cells would beparticularly susceptible to destruction by hemodynamic shear forces, ascompared to naturally circulating cells (i.e. red blood cells andleukocytes). One early study examined death of B16 melanoma cellssubjected to shear stress using a viscometer [Brooks, D. E., Thebiorheology of tumor cells. Biorheology, 1984. 21(1-2): p. 85-91.]. Thisreport showed dose-dependent killing of cells, however, the earliestviability time points analyzed were after one hour of shear stressexposure.

SUMMARY

Methods for purifying viable cancerous epithelial cells in an in vitrofluid preparation comprising viable cancerous epithelial cells, viablenormal epithelial cells, and extracellular calcium are provided. In someembodiments, the methods comprise: applying an initial pulse of fluidshear stress to the preparation, wherein the initial pulse of fluidshear stress induces a fluid shear stress resistance in the viablecancerous epithelial cells; and subsequently applying one or moreadditional pulses of fluid shear stress to the preparation comprisingthe fluid shear stress-resistant viable cancerous epithelial cells,whereby the ratio of viable cancerous epithelial cells to viable normalepithelial cells in the preparation is increased.

Also provided are methods for detecting cancerous epithelial cells in amammalian subject. In some embodiments, the methods comprise: obtaininga cell sample from the subject, the cell sample comprising cancerousepithelial cells and normal epithelial cells; forming a fluidpreparation comprising the cancerous epithelial cells, the normalepithelial cells and extracellular calcium; applying an initial pulse offluid shear stress to the preparation, wherein the initial pulse offluid shear stress induces a fluid shear stress resistance in thecancerous epithelial cells; subsequently applying one or more additionalpulses of fluid shear stress to the preparation comprising the fluidshear stress-resistant viable cancerous epithelial cells, whereby theratio of viable cancerous epithelial cells to viable normal epithelialcells in the preparation is increased; and subsequently measuring theamount of viable cancerous epithelial cells in the preparation.

Still further provided are methods for preparing a fluid preparationcomprising circulating tumor cells for a prognostic assay. In someembodiments, the methods comprise: obtaining a blood sample from thesubject, the blood sample comprising circulating tumor cells and normalepithelial cells; forming a fluid preparation comprising the circulatingtumor cells, the normal epithelial cells and extracellular calcium;applying an initial pulse of fluid shear stress to the fluidpreparation, wherein the initial pulse of fluid shear stress induces afluid shear stress resistance in the circulating tumor cells;subsequently applying one or more additional pulses of fluid shearstress to the fluid preparation, whereby the ratio of circulating tumorcells to viable normal epithelial cells in the preparation is increased;and subsequently conducting an assay on the preparation, the assayproviding a cancer prognosis for the subject based on the viablecancerous epithelial cells.

BRIEF DESCRIPTION OF THE DRAWINGS

Illustrative embodiments of the invention will hereafter be describedwith reference to the accompanying drawings.

FIGS. 1A-D show that an in vitro model of fluid shear stress inducescell death in a dose-dependent fashion. Suspensions of PC-3 cells weresubjected to repeated shear stress at increasing flow rates,corresponding to a range in shear forces from 509-6.36×10³dyn/cm² (seeTable 1), and monitored for changes in viability. Survival isrepresented as percent viability of non-shear treated cells which areheld in suspension for the duration of the assay. FIG. 1A: Endpointviability after 10 passages at indicated flow rate. (***, p<0.001 vs. 0control. One way ANOVA, Bonferroni's post-test; for each flow rate, n=5using syringe pump) FIG. 1B: Survival over repeated passages at 20 and250 μL/sec. (**p<0.01, *** p<0.001 vs. 20 μL/sec. Repeated measuresANOVA, Bonferroni's post-test; for each flow rate n=5 using syringepump). FIG. 1C: After 10 passages at 250 μL/sec, cell viabilityquantified via BLI was compared using WST-1 assay and clonogenic plating(Using one-way ANOVA, there are not significant differences between theresults obtained from these three methods; for each assay n=4 usingmanual method). FIG. 1D: To test the effects of culture conditions onshear stress survival, PC-3 cells were prepared under the followingconditions: “5×10⁴ vs. 5×10⁵ cells/mL”, from cells grown to 70%confluence; “low vs. medium vs. high confluence”, from cells grown to20-30%, 50-60%, or 100% confluence, respectively, prior to suspension at5×10⁵ cells/mL; “fresh vs. sheared”, trypsinized cells, grown to ˜70%confluence, were suspended in “fresh” media or were suspended in“sheared” media (cell-free media collected from cells sheared ten timesat 250 μL/sec); “versene”, cells were suspended non-enzymatically to5×10⁴cells/mL and compared to cells prepared similarly using trypsin.(No statistically significant differences using one-way ANOVA, Allexperiments n=4 using manual method).

FIGS. 2A-D show that carcinoma cells of various histological originsexhibit unique resistance to fluid shear stress. FIG. 2A: A panel oftransformed and normal epithelial and blood cells was compared forsurvival after 10 passages of fluid shear stress at 250 μL/sec. Cancercell lines exhibited robust resistance over other epithelial cell types.***, p<0.001 vs. all cancer cell lines; #, p<0.001 vs. RWPE-1;

, p<0.001 vs. all non-blood cells (one way ANOVA, Bonferroni post testsn=3 for blood cells by syringe pump method, n=6 for all other linesusing manual method). FIG. 2B: The viability of all cells in A at everyother passage. FIG. 2C: The rate of cell death per passage of allepithelial cell types represented in A. Note the rate of cancer celldeath significantly reduces and stabilizes after the second passage,whereas non-transformed cells remain constant. While primary cellsappear biphasic, they lose nearly all viability over the first twopassages. ***, p<0.001 vs. passages 1 to 2 of cancer cells; # p<0.05 vs.passages 1 to 2 of primary cells (one way ANOVA, Bonferroni post tests).FIG. 2D: To control for detachment-induced cell death, PC-3 and primarycells were held in suspension at room temperature without shear stresstreatment for up to one hour. Loss of viability due to detachment overthe first 30 minutes is insignificant by one-way ANOVA, n=5 for eachcell line).

FIG. 3 shows that shear stress resistance in carcinoma cells istransient and inducible. After 10 passages of fluid shear stress at 250μL/sec, surviving PC-C, PC-3 adrenal gland-derivative, and B16.f0 cellswere allowed to recover in culture for 24-48 hours. These survivors werethen compared for shear stress resistance in parallel with thecorresponding shear stress-naïve control cells. Subculturing survivingcells did not enrich for shear stress resistance at 250 μL/sec (nosignificant differences by one-way ANOVA, n=3 for each cell line usingmanual method).

FIGS. 4A-D show that selective and diminishing accumulation ofmembrane-impermeable dye in PC-3 cells exposed to shear stress reflectschanges in cellular membrane integrity and induced resistance to shearforces. PC-3 cells subjected to shear stress in the presence or absenceof propidium iodide (PI) were collected after each passage for flowcytometric analysis. PI was added to the cell suspension either prior toshear passage 1 (p1), 6 (p6), 8 (p8), or 10 (p10). Viable cells (P1+P2gated), defined by cell size, shape, and density (forward scatter andside scatter), were evaluated for PI positivity. When exposed to PIconstantly (FIG. 4A, upper right), p1 resulted in 7.28% of thepopulation of viable cells accumulating PI intracellularly, as comparedto non-sheared cells (0.65%, FIG. 4A, upper left). With sequentialshearing, the p10 sample resulted in a final accumulation of PI in36.73% in the viable cell population (FIG. 4A, lower left). In contrast,when PI was introduced to the cells prior to p10 (FIG. 4A, lower right),only 4.22% of the cell population accumulated PI. FIG. 4B: To confirmthat the P1+P2 gate represented only viable cells, and to eliminate thepossibility that PI⁺-dead cells were contributing to our PI⁺ gate, thevital stain Calcein AM was used and confirmed that the P1+P2 gate waspredominantly constituted by viable cells (p1 99.8%, p10 99.2%). FIG.4C: When PI was added prior to passage 6 (pre-6), 8 (pre-8), or 10(pre-10), less of the viable population of cells accumulated the dyewith the first passage with PI. With subsequent passages displayed aplateau of accumulation, much as see in the late passages in constantpresence of Pl. This reflects a shear force-resistant phenotype thatexists after 1-2 shear passages in a subset of remaining viable cells,and explains the biphasic change in cell viability observed over 10passages (FIG. 2B). (*p<0.05 vs. 1constant; for each condition, n=8using syringe pump).

FIGS. 5A-B show that shear stress-induced cancer cell membrane repairrequires presence of extracellular calcium. FIG. 5A: PC-3 cellssuspended in complete medium, calcium-free PBS, or PBS plus calcium(129.4 μg/mL final concentration) were subjected to shear stress at 250μL/sec. The absence of calcium leads to significantly increased celldeath whereas cells suspended in calcium-supplemented PBS exhibit cellsurvival similar to that of cells in media. FIG. 5B: The same experimentas described in FIG. 5A was performed at 20 μL/sec. In complete medium,this flow rate induces little loss of viability in PC-3 cells; howeverin calcium-free PBS, cell death is rapid and linear. *p<0.05, **p<0.01,*** p<0.001 vs. complete media, Repeated measures ANOVA, Bonferroni'spost-test; for each condition, n=6 using syringe pump).

FIGS. 6A-B show that induced shear stress resistance requires actinpolymerization. FIG. 6A: PC-3 cells were treated with 20 nMcytochalasin-D for one hour before exposure to the shear stressprotocol. FIG. 6B: MDA.MB.231 cells were treated with 20 nMcytochalasin-D for one hour before exposure to the shear stressprotocol. Cytochalasin-D treated cells are much more susceptible toshear stress than DMSO control cell suspensions. **p<0.01, *** p<0.001vs. DMSO control (one way ANOVA, Bonferroni post test; for eachcondition, n=4 using manual method).

FIG. 7: Clonogenic survival correlates with bioluminescence imagingviability data. PC-3, TEM4-18, and PrEC cells which had been subjectedto 0, 2, or 10 passages at 250 μL/sec (6.36×10³dyn/cm²) were plated atclonal density. Colonies resulting from live, single cells were stainedand scored. Data shown on graph is the average of three independentshear treatments and subsequent plating assays. This data correlateswell with viability data derived via bioluminescence imaging. n=3 foreach condition, done using manual method.

FIG. 8: Comparison of cancer cell death under needle and syringe andrheometer fluid shear stress techniques. PC-3 cell viability data shownin FIG. 1A (checkered bars) is compared to PC-3 cells treated atescalating doses of shear stress using a rheometer. All data wereacquired using BLI and rheometer experiments were done three times.

FIG. 9: Response of PC-3 cells to shear stress is similar over a rangeof time post-suspension. An aliquot of freshly suspended PC-3 cells wasanalyzed for shear stress survival (1^(st)). Once this first protocolwas finished, another aliquot of the PC-3 stock was subjected to theprotocol (2^(nd)). One hour after suspension, a final round of shearprotocol was performed (3^(rd)). Regardless of time since preparing thecell suspension, the response of PC-3 cells to shear stress was similar.Survival is represented as percent viability of non-shear treated cellswhich are held in suspension for the duration of the assay (for eachtime period post suspension, n=3 using manual method).

FIGS. 10A-B show a shear stress analysis of a broad panel of cells.Cancer cells derived from various epithelial tissues, as well ashematogenous origin were analyzed for survival over ten passages ofshear stress at 250 μL/sec. FIG. 10A displays the endpoint viability andFIG. 10B displays the viability over repeated passages. For each cellline, survival is represented as percent viability of non-shear treatedcells which are held in suspension for the duration of the assay. Celllines obtained from experimental metastases in mice were included forPC-3 (AD, adrenal gland; LD, liver), MDA.MB.231 (LuD, lung), B16f0(B16f10, 10-times serially passaged intravenously to lung), and 22Rv1(BD, long bone). These in vivo derivatives do not exhibit increasedshear stress resistance. For each cell line experiments were done atleast three times using the pump method.

FIG. 11: Shear stress resistance response specifically requires calcium.PC-3 cells suspended in complete medium, calcium-free PBS, or PBS pluseither calcium, magnesium, or barium (88 mM final concentration,respectively) were subjected to shear stress at 250 μL/sec. In PBS,shear stress induced is greatly enhanced. Only addition of calcium toPBS rescues the shear stress resistance phenotype. Currently, n=1 foreach condition using syringe pump.

FIG. 12: a schematic scale illustration of a PC-3 cell subjected to FSSin a conduit.

FIGS. 13A-B show primary cell survival at 20 μL/sec. PrEC and HMECsuspensions were subjected to ten passages of FSS at 20 μL/sec. FIG.13A: Viability at every other passage. FIG. 13B: Comparison of PrEC,HMEC, and PC-3 survival after ten passages at 20 μL/sec.

FIGS. 14A-B show enrichment of malignant cells from a mixed cellsuspension by fluid shear stress. FIG. 14A: Suspensions of PC-3 and PrECwere labeled with calcein AM (CAM) and cytotracker orange, respectively,and mixed ˜1:1. Before (P.0) and after ten passages (P.10) of FSS,10,000 fluorescent events were counted using flow cytometry. FIG. 14B:25 μL of mixed (PC-3:PrEC) cell suspension was plated into collagen1-coated 8-well chamber slides before (P.0) and after ten passages(P.10) of FSS. These cells were allowed to adhere overnight and werethen fixed in 4% paraformaldehyde for 10 minutes. The ratio of calceinAM positive to negative cells is shown.

FIG. 15A shows the effect of FSS (at 250 μL/s) on wild type primaryhuman prostate epithelial cells (PrEC), immortalized PrEC (LH), andMyc/PI3K (LHMK) or Ras (LHSR) transformed PrEC; FIG. 15B shows a graphshowing that R545 melanoma cells (derived from Tyr/Tet-Ras INK4a^(−/−)mice) express H-Rasv12 in a doxycycline-dependent manner.

FIG. 16A shows a graph showing cell viability for PC-3 cells suspendedin DMEM/F12, 10% FBS in the presence or absence of 20 mM HEPES (avg. pHat room temperature: 7.3 vs. 7.7, respectively). Also shown is the cellviability data for the PC-3 cells suspended in regular media sheared at37° C. in a walk-in incubator. FIG. 16B shows a graph showing cellviability data for sheared primary cells (HMEC, n=2 and PrEC, n=5experiments using pump method) at 37° C. and at room temperature.

FIG. 17: Graph showing viability for cells freshly isolated from murineprostates as a function of repeated exposure to fluid shear stresses.

DETAILED DESCRIPTION

Methods for isolating viable cancer cells from a sample that comprises amixture of cancerous cells and normal (non-cancerous) cells areprovided. Also provided are methods for detecting cancerous cells in asubject and methods for preparing patient samples, such as bloodsamples, for prognostic or diagnostic assays predictions based on thedetection and/or concentration of cancerous cells in the sample. Amotivation behind the methods may be attributed, at least in part, tothe discovery that fluid shear stress can be used to selectively killnormal cells in a preparation that includes both normal and cancerouscells. Moreover, in addition to the ability to selectively kill normalcells relative to cancerous cells, the present methods may provide theability to selectively kill cancerous cells based on their level ofmetastatic potential. As such, the methods can be used to enrich apreparation with more aggressive and, thus, more prognostically valuablecancer cells.

In the methods, a liquid preparation comprising a suspension of cancercells is repeatedly exposed to fluid shear stresses. For example, thepreparation can be passed through a conduit, desirably at asubstantially constant flow rate, multiple times, whereby the repeatedexposure to the resulting fluid shear stresses imparts an increasedresistance to fluid shear stress to the cancer cells. The normal cellsin the preparation do not experience an increase in their resistance tofluid shear stress or experience a lower increase than the cancerouscells and, therefore, are susceptible to magnitudes of shear stress muchlower than those required to kill cancer cells. The resistance to shearstress is not believed to be a stable, genetic trait, but rather atransient, adaptive response that is able to develop in the presence ofextracellular calcium.

In one embodiment, the methods provide for the purification of viablecancerous cells in a fluid preparation comprising a mixture of viablecancerous cells, viable normal cells, and extracellular calcium. At theoutset, the preparation can be characterized by an initial ratio ofviable cancerous cells to viable normal cells. In this embodiment, themethod comprises the steps of applying an initial pulse of fluid shearstress to the preparation, wherein the initial pulse of fluid shearstress induces a fluid shear stress resistance in the viable cancerouscells. Subsequently one or more additional pulses of fluid shear stressare applied to the preparation, which now comprises the fluid shearstress-resistant viable cancerous cells. As a result of the applicationof the fluid shear stresses to the preparation, the ratio of viablecancerous cells to viable normal cells in the preparation is increasedand the preparation is thereby purified. Optionally, the purifiedpreparation can further processed by separating the viable cells fromthe non-viable cells and/or by separating the cancerous cells from thenormal cells.

As a result of the present methods, the cancerous cells in a preparationmay be observed to exhibit a biphasic viability behavior, wherein afterthe initial resistance-inducing pulse, the observed loss in cellviability is significantly reduced for subsequent pulses.

The present methods can further provide for the detection andquantification of cancerous cells in a subject, such as a human patient,with cancer. In one embodiment of such a method, a cell samplecomprising cancerous cells and normal cells is obtained from the subjectand a fluid preparation comprising the cancerous and normal cells in thepresence of extracellular calcium is prepared. Here again, the methodcomprises the steps of applying an initial pulse of fluid shear stressto the preparation, wherein the initial pulse of fluid shear stressinduces a fluid shear stress resistance in the viable cancerous cells.Subsequently one or more additional pulses of fluid shear stress areapplied to the preparation, which now comprises the fluid shearstress-resistant viable cancerous cells. As a result of the applicationof the fluid shear stresses to the preparation, the ratio of viablecancerous cells to viable normal cells in the preparation is increasedand the preparation is thereby purified. The amount (e.g.,concentration) of viable cancerous cells in the purified preparation isthen measured.

The results of the measurement can be used, for example, in diagnosticor prognostic assays to assess the likely health outcome for the subjectfrom whom the cell sample was taken. Thus, the present methods provide arelatively simple, relatively low-cost method for providing preparationsthat are enriched in viable cancerous cells relative to normal(non-cancerous cells) and, as such, are well-suited for use in clinicalassays. For example, the results of measurements taken on purifiedpreparations can be correlated to the likelihood of relapse ormetastasis in a patient.

By way of illustration, the present methods can be used to purify apreparation comprising a blood sample comprising CTCs for a patient withbreast cancer prior to surgery and correlating higher numbers ofcirculating viable cancer cells with an increased likelihood of breastcancer relapse. Similarly, the present methods can be used to purify apreparation comprising CTCs from a patient with non-small cell lungcarcinoma after said patient has had surgery for removal of thecarcinoma and correlating higher numbers of circulating viable cancercells with an increased likelihood of cancer relapse.

The types cancerous and normal epithelial cells that can be subjected tothe present methods cover a broad range of mammalian (e.g., human)cells. The preparations comprising the cells can comprise, for example,blood samples, or extracts from blood samples, containing CTCs.Alternatively, the preparation comprising the cells can be prepared fromtissue samples. For example, the preparations can comprise suspensionsof tumor cells obtained from the biopsy of a solid tumor.

The methods are general and may be conducted on a preparation to isolatecancer cells of any type. Examples of the types of cells that may bepresent in the preparations include normal and cancerous prostate cells,breast cells, pancreatic cells, colon cells, ovarian cells, plasmacells, lung cells, adrenal cells, liver cells, lymphocyte cells andcombinations thereof.

The preparations comprising the cancerous and normal cells can besubjected to pulses of fluid shear stress by passing them through aconduit in, for example, a microfluidic device. Examples of suitableconduits can include, for example, a capillary tube or a needle. Thepreparations are desirably passed through the conduit at a constant orsubstantially constant rate. This can be accomplished, for example,using a mechanical pump or by hand—as in the case of a syringe.

As discussed above, an initial pulse of fluid shear stress is applied tothe preparation in order to induce an increased resistance to fluidshear stress-induced cell death to the cancerous cells that survive theinitial pulse or pulses. By way of clarification, the term ‘initialpulse’ is not intended to imply that only a single ‘initial pulse’ canbe applied. Instead the term ‘initial pulse’ refers to a pulse that isused to provide a cell that has been subjected to that pulse with anincreased resistance to subsequently applied fluid shear stresses. Thus,an initial pulse is a fluid shear stress resistance-inducing pulse. Inthe present methods more than one ‘initial pulse’ may be necessary ordesirable. Therefore, the phrase “applying an initial shear stress to apreparation” does not preclude the possibility of applying more than oneinitial shear stress to the preparation. Further, in some embodiments,the initial pulse (or pulses) and the subsequent additional pulses areapplied in vitro. However, if the sample from which the preparation isprepared has already been subjected to an stress resistance-inducingpulse in vivo by virtue of its having experienced a shear stress duringcirculation and the preparation is processed sufficiently rapidly (i.e.,rapidly enough that the stress resistance has not subsided), then theinitial pulse may actually be an in vivo pulse.

Both the strength (i.e., the level of shear stress) and the duration ofthe pulse will affect cell viability in the preparation. Therefore, itis advantageous to select a pulse strength and duration that aresufficient to induce an increased fluid shear stress resistance withoutreducing the concentration of viable cancerous cells in the preparationto a level that is unacceptable for its intended purpose.

For the purposes of this disclosure, fluid shear stress can be measuredby the wall shear stress experienced at the wall of a conduit throughwhich a fluid preparation is passed, calculated using Poiseuille'sequation, as described in Example 1, below.

The magnitude of the fluid shear stresses to which the preparation issubjected can vary over a wide range, which includes high and/orsupra-physiologic levels (i.e., levels of fluid shear stress that arehigher than would be experienced in human circulation). By way ofillustration, in some embodiments, the magnitude of the initial pulse(s)of fluid shear stress is in the range from about 300 to about 6500dyn/cm². This includes embodiments in which the magnitude of the initialpulse(s) of fluid shear stress is in the range from about 500 to about6500 dyn/cm² and further includes embodiments in which the magnitude ofthe initial pulse(s) of fluid shear stress is in the range from about700 to about 6000 dyn/cm².

Like the magnitude of the pulses of fluid shear stress, the duration ofthe pulses of fluid shear stress used in the present methods can varyover a considerable range. In general, the duration should be longenough to impart a fluid shear stress-resistance to the cancerous cellsin the preparation without reducing the number of viable cancerous cellsin the preparation to an unacceptably-low value. This can beaccomplished with very short pulses, which allows the methods to becarried out rapidly. By way of illustration, in some embodiments, theinitial pulse(s) of fluid shear stress has a duration of no greater thanabout 20 msec. This includes embodiments in which the initial pulse(s)of fluid shear stress has a duration of no greater than about 15 msec.For example, in some embodiments, the initial pulse(s) of fluid shearstress has a duration in the range from about 0.5 msec to about 15 msec.

The one or more additional pulses of fluid shear stress applied to thepreparation after the one or more resistance-inducing initial pulses canbe used to increase the ratio of viable cancerous cells to viable normalcells in the preparation. Because the fluid shear stress resistanceimparted by the initial pulses can diminish over time, the additionalpulses should be applied prior to the loss of the induced fluid shearstress-resistance. Thus, in some embodiments, at least the first of theone or more additional pulses are applied within no more than one hourafter the initial pulse. This includes embodiments in which at least thefirst of the one or more additional pulses are applied within no morethan 15 minutes after the initial pulse and further includes embodimentsin which at least the first of the one or more additional pulses areapplied within no more than five minutes after the initial pulse. Insome embodiments all of the one or more additional pulses are appliedwithin these time windows.

The magnitude and duration of the additional pulses of fluid shearstress can be the same as, similar to, or different from those of theinitial pulse of fluid shear stress. Thus, in some embodiments, theadditional pulse(s) of fluid shear stress is in the range from about 300to about 6500 dyn/cm². This includes embodiments in which the additionalpulse(s) of fluid shear stress is in the range from about 500 to about6500 dyn/cm² and further includes embodiments in which the additionalpulse(s) of fluid shear stress is in the range from about 700 to about6000 dyn/cm². In some embodiments, the initial pulse applies a lowerlevel of fluid shear stress that than the additional pulses. Forexample, the initial pulses may apply a physiologic level of fluid shearstress, while the one or more additional pulses apply asupra-physiologic level of fluid shear stress.

In some embodiments, the additional pulse(s) of fluid shear stress has aduration of no greater than about 20 msec. This includes embodiments inwhich the additional pulse of fluid shear stress has a duration of nogreater than about 15 msec. For example, in some embodiments, theadditional pulse(s) of fluid shear stress have a duration in the rangefrom about 0.5 msec to about 15 msec.

The number of additional pulses can be selected to provide a desiredratio or maximized ratio of viable cancerous cells to viable normalcells in the preparation. By way of illustration, in variousembodiments, the methods can use ≦100, ≦50, ≦20, ≦10 or ≦5 additionalpulses. For example, in some embodiments, 2-50, 2-20 or 2-10 additionalpulses are applied.

The magnitude of the initial and additional pulses of fluid shear stresscan be selected such that they provide at least a minimum acceptablelevel of cancerous cell viability after processing and/or such that theyprovide a desired enhancement in the ratio of cancerous to normal cellsin the preparation. For example, lower fluid shear stresses (e.g., ≦1000dyn/cm²) can be used to retain a higher overall cell viability, whereashigher fluid shear stresses (e.g., ≧400 dyn/cm²) can be used to providea higher degree of selective killing of normal cells and, therefore, agreater ratio of cancerous to normal cells.

Generally, the duration of the pulses, the magnitude of the pulses andthe number of pulses can be tailored to provide a desired ratio ofviable cancerous cells to viable normal cells, or to provide at least aminimum cancerous cell viability, as measured substantially immediatelyafter the additional pulse sequence has been carried out. Thus, by wayof illustration, these variable can be selected to provide a ratio ofviable cancerous cells to normal cells of at least 2:1, at least 3:1 orat least 10:1 and/or to provide a cancerous cell viability of at least30%, at least 50% or at least 80%.

Once a fluid preparation has been purified using the present methods,the concentration of viable cancerous cells can be measured and,optionally, quantified. Quantification can entail, for example, countingthe number of cells using a method such as Fluorescence Activated CellSorting (FACS), or measuring the percent cell viability. Variousillustrative methods for measuring cell viability are presented in theexamples that follow. For the purposes of this disclosure, the percentof cell viability in a cell sample is determined relative to a controlsample composed of the same types of cells in the same preparation thathas been maintained, unexposed to fluid shear stresses, for the durationof the fluid shear stress-based purification process. If the reductionin cell viability resulting from the purification process is to bequantified, it is desirable to measure the percent viabilitysubstantially immediately after the purification process is completed.That is, before natural cell death occurs to a degree significant enoughto alter the measurement.

The purification methods have the capability to substantially increasethe ratio of viable cancerous cells to viable normal cells in apreparation comprising both. For example, in some embodiments, the ratioof viable cancerous cells to viable normal cells in the purifiedpreparation is at least doubled relative to the ratio prior topurification. This includes embodiments in which the ratio of viablecancerous cells to viable normal cells in the purified preparation is atleast tripled relative to the ratio prior to purification and furtherincludes embodiments in which the ratio is increased by at least afactor of five, at least a factor of 8 or at least a factor of 10.

The methods also have the capability of significantly reducing thepercent of viable normal cells in a in a preparation withoutsubstantially reducing the percent of viable cancerous cells. Forexample, in some embodiments, the methods provide a purifiedpreparation, wherein the percent viability for the normal cells is nogreater than 5% and the percent viability for the cancerous cells is atleast 40%. This includes embodiments of the methods that provide apurified preparation, wherein the percent viability for the normal cellsis no greater than 10% and the percent viability for the cancerous cellsis at least 70%.

The devices that have been described herein for carrying out the methodsare also provided. In one embodiment, a device for applying fluid shearstress to a fluid preparation comprises a conduit configured to containthe fluid preparation, a pump configured to pass the fluid preparationthrough the conduit, and a flow controller configured to control therate of flow of the fluid preparation through the conduit, wherein thedevice is configured to apply one or more pulses of fluid shear stressto the fluid preparation via the pump and flow controller, the one ormore pulses sufficient to increase the ratio of cancerous cells tonormal cells in the fluid preparation. In some embodiments, the deviceis configured to apply an initial pulse of fluid shear stress sufficientto induce a fluid shear stress resistance in cancerous cells in thefluid preparation and to subsequently apply one or more additionalpulses of fluid shear stress to the fluid preparation. In someembodiments, the device is configured to pass the fluid preparationthrough the conduit at a substantially constant flow rate. Any of thefluid preparations described herein may be used, e.g., a fluidpreparation comprising cancerous epithelial cells, normal epithelialcells and extracellular calcium. The device may be configured to applyany number of pulses of fluid shear stress (e.g., initial pulses,additional pulses) having any of the magnitudes, durations and timingsdescribed herein. The device may be configured to apply pulses of fluidshear stress sufficient to provide any of the ratios of cancerous cellsto normal cells and/or levels of cancerous cell viability and/or levelsof normal cell viability described herein.

EXAMPLES

The following examples illustrate the usefulness of the present methodsfor detecting, quantifying, purifying and/or isolating cancerousepithelial cells from preparations comprising circulating tumor cellsand normal cells. The examples present possible theories and hypothesisin support of the present methods. However, the inventors do not intendto be bound to any particular theory of the inventions, and the theoriespresented here are not intended to restrict the claimed subject matter.

Example 1

This example illustrates the use of the present methods to purify apreparation prepared from a blood sample comprising CTCs.

Materials and Methods

Cell Lines

All cancer cell lines were obtained from the ATCC and were transducedwith an integrating retrovirus encoding firefly luciferase under controlof the CMV promoter (pGEM, Promega). Cells were grown in theATCC-recommended media supplemented with 10% fetal bovine serum and 1%non-essential amino acids. For maintenance of the retrovirus-geneexpression, all cells containing this construct were maintained in 200μg/mL Genetecin (Invitrogen). Primary prostatic and mammary epithelialcells were obtained from Clontech and were cultured in their commercialdefined media. For all experiments, cells were grown to ˜75% confluenceand harvested by trypsinization following neutralization and suspensionin complete medium.

Human and Mouse Blood

To obtain whole human blood, fresh leuko-reduction cones were collectedfrom the University of Iowa Hospitals and Clinics DeGowan Blood Center.Cones were flushed in direction of filtration with normal saline (0.99%NaCl) to reduce red blood cell (RBC) content. Diluted RBCS werecollected and brought to a concentration of 5×10⁵ cells/mL and used inshear stress experiments. To isolate leukocytes, cones were then elutedin the direction opposite of filtration using 50 mL ACK buffer (150 mMNH₄Cl₄, 10 mM KHCl₃, 0.10 mM ETDA, pH 7.4), which osmotically lysesremaining RBCS. After 15 minutes of incubation at room temperature,cells were centrifuged at 100RCF for 5 minutes, resuspended in 1 mLPBS+xM calcein AM viability dye (Invitrogen), and incubated for an 15minutes at room temperature. 9 mL of ACK buffer was added to this cellsuspension, centrifuged once more as above, and brought to a finalconcentration of 5×10⁵ cells/mL in DMEM (Gibco) without supplements.

For whole mouse blood, sub-mandibular bleeds were performed on adultBI/6 mice. Blood was collected in EDTA-treated tubes (BD biosciences)and diluted using normal saline to a concentration of 5×10⁵ cells/mLprior to shear stress treatment.

Cell Size Analysis

Cells were suspended to a concentration of 5×10⁵cell/mL and analyzed ona Coulter Counter (Beckman Coulter) at a 1:100 dilution in Isoton II(Beckman Coulter). Size analysis was performed using Z2 Acucomp software(Beckman Coulter). Gates were set to exclude cellular debris andaggregates following the manufacturer's instructions. At least threeseparate cultures for each cell line were counted and sized intriplicate. Data was plotted using Prism Graph Pad software and celllines were compared based on mean cell volume. Paired, two-tailedt-tests were used for analysis of statistical significance.

Shear Stress Equations

Wall shear stress was calculated using Poiseuille's equation, T=4Qη/πr3[Davies, P. F., Hemodynamic shear stress and the endothelium incardiovascular pathophysiology. Nat Clin Pract Cardiovasc Med, 2009.6(1): p. 16-26.], where T is shear stress in dyn/cm²; Q is flow rate incm³/s; η is the viscosity of the medium (0.01 dyn/cm/s for culturemedia); and r is the radius of the needle (30GA average internal radius=7.94×10⁻³cm). FIG. 12 is a diagram of a fluid moving through a conduitand illustrates a cell subject to a gradient of shear stress, withmagnitude depending on its position relative to the axis of the conduit.

For calculating the minimum shear stress experienced by a given cell,the equation T=ΔPr/2L was used, where ΔP is the change in pressure, r isthe radius of the cell, and L is the length of the needle (1.27 cm). Theassumption was made that cells were flowing along the axis of the needlein single file; thus keeping everything constant, the cell radius willdetermine the magnitude of shear force encountered.

Reynolds number was calculated to assume laminar flow using the equationR_(e)=ρvD/η where ρ is the density of the culture media (0.998 g/cm³), vis the velocity of flow, D is the diameter of the needle, and η is theviscosity of the medium. For the 20 μL/sec (low) flow rate, R_(e) is159.58; for the 250 μL/sec (high) flow rate, R_(e) is 1998. Thisindicates that all flow rates in the shear stress model were laminar.

Shear Stress Models

5 mL of a 5×10⁵ cell/mL suspension was placed into 10mL glass beaker(Pyrex) and loaded into a 5mL syringe (BD Biosciences #309603) by slowlydrawing the cells into the syringe manually. A 30 gauge needle (BDBiosciences #305106) was then attached to the syringe and cells werepushed through at a constant flow rate by one of two methods:

Syringe Pump: A Harvard Apparatus PHD-2000 Infuse/Withdraw syringe pumpwas calibrated for the syringes being used and set to the appropriateflow rate (see Table I). After securing the syringe to the pump housing,a 6-inch small bore extension set (Smiths Medical MX448L) was used toconnect the syringe and needle. The 10 mL beaker was secured at a45-degree angle and the needle rested gently (without damaging thebevel) at the bottom of the beaker. The pump is then turned on and thecell suspension was collected into the beaker. The bore extension, stillconnected to the needle, was then removed from the syringe, and thesyringe was removed from the pump. The cell suspension was re-loadedinto the syringe as described above. In a standard assay, this processwas repeated until cells have been passaged a total of ten times(control experiments were taken out to 15 passages). After each passage,100 μL aliquots of cell suspension were removed from the glass beaker induplicate for viability assays (described below). For non-shear stresstreated control, cells which have been in suspension for the entireduration of the assay were aliquotted and treated as 100% viabilitycontrols.

250 μL/sec flow rate: This is the most commonly used flow ratethroughout this example. To facilitate throughput, some experiments at250 μL/sec were done manually. Here, cell suspensions were prepared asabove with one exception: the small bore extension was not used, ratherthe needle was directly secured to the syringe. Suspensions were pushedthrough the needle by hand and collected into a 10 mL glass beaker. Tocontrol the flow rate using this approach, a timer was zeroed beforeeach needle passage and the time taken to push a given volume throughthe needle was measured. At each passage, the volume in milliliters wasdivided by the time in seconds to give flow rate in mL/sec. Data forfigures was only obtained from shear stress assays in which the averageflow rate over 10 passages was ±10 μL/sec of the targeted 250 μL/sec. Asdata in FIGS. 1A-D indicate, fluctuations within 10 μL/sec at this flowrate should not lead to significantly different viability results.Further, to assure reproducibility, most assays were repeated ten timesor more.

Cell Viability Assays

Bioluminescence Imaging (BLI): 100 μL aliquots of shear stress-treatedcells or control cells (those sitting in suspension through the durationof shear treatment) were loaded into a black 96 well plate (Costar) induplicate. Each well was then diluted to 200 μL at final concentrationof 150 μg/mL D-luciferin (Promega) using a multichannel pipette. Plateswere incubated for 5 minutes at room temperature and then imaged for 5minutes in an IVIS-100 (Xenogen). Bioluminescence measurements werecollected using Living Image 2.50.1 software (Igor Pro). The photon fluxof shear treated cells was divided by that of control cells to give %viability. All figures with BLI-derived viability data represent theaverage of at least 3 experiments.

WST-1 Viability assay: For primary cells and cell lines lackingluciferase-expression, cell viability was measured with(4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3 benzenedisulfonate (WST-1, Roche Applied Science) as directed. As indicated inFIGS. 1A-D, WST-1 and BLI results were compared in parallel, showingagreement between the two methods.

Clonogenic plating for quantification of cell viability: To support cellviability data acquired acutely using BLI and WST-1, clonal survivalplating was performed. Here, 5 μL of a 1:10 dilution of control or shearstress-treated cells was plated into 10cm plate in 10 mL of theappropriate complete culture media plus pen/strep. This volume containsan estimated 250 live cells prior to shear stress treatment (5×10⁶cell/mL×0.5×10⁻⁴ mL=250 cells). Plates were incubated until visiblecolonies formed from single cells (depending on the cell line, thistakes 1-2 weeks). Colonies were stained overnight with PBS containing0.01% crystal violet and 0.02% citric acid, washed with distilled water,and counted on a light box. The number of colonies from shear treatedsuspensions was divided by that of control plates to give percentsurvival. Graphs derived using this approach represent the average ofthree experiments or more.

Flow cytometry for quantitative analysis of cell viability: Thistechnique was used for leukocytes obtained from whole blood. Leukocytes,prepared as described above, were subjected to shear stress for tenpassages. Before shearing and after each passage, 300 μL aliquots wereplaced in XXmL FACS tubes (BD Biosciences) in duplicate. 100 μL of DMEMcontaining 1×10⁶ counting beads/mL (Caltag Laboratories) was added toeach tube, as well as propidium iodide to a final concentration of 0.5μg/mL. Single cells were gated on forward and side scatter, on aBectonDickenson LSR. Stop gates were set to 5,000 counting beads toassure consistency across samples. The ratio of live (propidiumiodide-negative, calcein-AM-positive) cells to counting beads at eachpassage was compared to non-shear stress-treated controls to providepercent viability.

Flow Cytometry Analysis of Propidium Iodide Uptake

200 μL of cell solution was taken for each sample and added to 200 μL ofcomplete culture medium in 5 mL polystyrene FACS tubes (BD Biosciences).Propidium iodide (final concentration of 0.5 μg/mL) was added eitherbefore the first, sixth, eighth, or tenth shear passages. Cells wereanalyzed on a BectonDickenson LSR with Violet. Single cells were gatedby forward and side scatter, consistent with viability, and evaluatedfor PI and/or Calcein AM positivity.

Statistical Analyses

For statistical analysis of cell size and endpoint shear stresssurvival, paired 2-tailed t-tests were used when comparing two celllines or flow rates. When comparing the endpoint survival of three ormore cell lines or flow rates, one-way ANOVA followed by Bonferroni posttests were performed. When comparing shear stress survival of multiplecell lines over repeated passages, repeated measures ANOVA followed byBonferroni post tests were used.

Results

In Vitro Model of Fluid Shear Stress Induces Cell Death in aDose-Dependent Fashion.

To directly test the effect of fluid shear stress on cancer cells, anexperimental protocol involving the repeated passage of cell suspensionsthrough a 30 gauge needle (150 μm internal diameter) was used. Byholding a constant flow rate, the magnitude shear stress experienced atthe wall of the needle was controlled. Changes in cell viabilityresulting from exposure to shear stress were closely monitoredthroughout the protocol using bioluminescence imaging (BLI). The rangeof wall shear stress targeted in the protocol (Table 1) encompassed highphysiological values estimated in the human vasculature, and beyond[Reneman, R. S. and A. P. Hoeks, Wall shear stress as measured in vivo:consequences for the design of the arterial system. Med Biol Eng Comput,2008. 46(5): p. 499-507., Schneider, S. W., et al., Shear-inducedunfolding triggers adhesion of von Willebrand factor fibers. Proc NatlAcad Sci U S A, 2007. 104(19): p. 7899-903.]. Using a Coulter Counter,the mean cell radius of PC-3 was determined to be 9.3 μm. Applying theshear stress equation, this radius was used to estimate the minimal(axial) shear forces PC-3 cells encounter in this model. These valuesare shown for each flow rate in Table 1.

Row Rate Wall Shear Stress Minimum Shear Time of passage (μL/sec)(dyn/cm2) Stress (dyn/cm2) (msec) 20 509 59.53 12.2 35 890 104.09 6.9750 1.271 148.65 4.88 100 2.543 297.43 2.44 150 3.815 446.20 1.62 2506.358 743.63 0.89

Using this protocol, the survival of the human prostatic carcinoma cellline, PC-3, was analyzed after 10 passages of fluid shear stress (FIG.1A) and revealed a dose-dependent sensitivity of these cells to shearstress. When analyzing the survival curves of PC-3 cells over repeatedshear stress passages (FIG. 1B) little loss of viability was observed atthe lowest flow rate and a biphasic loss of viability at the highestflow rate. The cell viability measurements acquired with BLI wereconfirmed by using other techniques to assess cell viability followingthe shear stress protocol. A WST-1 assay of mitochondrial function aswell as clonogenic survival of control and shear stress-treated PC-3cells were performed. Results from these other methods mirrored acutechanges in viability measurements obtained using BLI (FIG. 1C). FIG. 7shows a comparison of PC-3 clonogenic plating with cells used in laterfigures.

Since shear force experienced by individual cells in this experimentcould be influenced by neighboring cells in suspension, PC-3 survivalover a 10-fold range of cell concentration was evaluated. It was shownthat shear stress induced cell death was not affected by cellconcentrations between 5×10⁴ and 5×10⁵ cells/mL (FIG. 1D). Whether cellviability in the shear stress protocol is influenced by the confluence(growth phase) of cells prior to suspension was also investigated. PC-3cells were trypsinized at low and medium confluence, as well as thosewhich had just reached full confluence (but not over-grown) and similarshear stress survival was found (FIG. 1D). To show that cell viabilitywas not affected by material released by damaged or dead cells, freshcells were suspended in cell-cleared medium from shear stress-treatedcells. Cell viability of these suspensions was not different than cellsin fresh media after 10 passages at 250 μL/sec (FIG. 1D). In addition,it was shown that the viability of cells in the protocol was similarbetween those collected by trypsin and versene (non-enzymatic) treatment(FIG. 1D). Finally, conditions tested included needle length (FIG. 1D).Only under this final condition, where the time of exposure to FSS waseffectively doubled, was a significant difference in endpoint survivalnoted. (*p<0.05 vs all other conditions, one-way ANOVA, followed byBonferroni's post test. All experiments n=4 using pump method.)

Collectively, these data show that loss of cell viability increasesproportionally with the magnitude of shear stress in a mannerindependent of cell culture and suspension preparation.

To help validate the model, the viability of cells in FIG. 1A werecompared to PC-3 cells subjected to short pulses of shear force using arheometer. Over a range of shear stress magnitudes, this approach causeda similar amount of cell death as our needle and syringe system (FIG.8).

Carcinoma Cells of Various Histological Origins Exhibit UniqueResistance to Fluid Shear Stress

Next, the shear stress protocol was employed to test for differences insurvival between carcinoma cell lines derived from metastatic prostate(PC-3 and 22Rv1), breast (MDA.MB.231), and melanoma (B16f0).Surprisingly, only small, insignificant differences in the survival ofthese cancer cell lines at the 250 μL/sec flow rate (black bars in FIG.2A and FIG. 2B) was observed. Also included in this analysis were twoimmortalized, but non-transformed cell lines of the human prostate(PWR-1E and RWPE-1) as well as primary cells of the human breast andprostate (HMEC and PrEC, respectively). Much greater cell deaths weremeasured in these epithelial cell types versus all cancer cell lines(FIG. 2A and FIG. 2B). Freshly isolated mouse red blood cells (RBCS), onthe other hand, are robustly resistant to this level of shear stress(FIG. 2A and FIG. 2B). Similarly, freshly isolated human leukocytes alsodisplay great resistance to shear forces (FIG. 2A and FIG. 2B). Thisfinding underscores the unexpected ability of these transformedepithelial cells to survive these magnitudes of shear stress.

As mentioned above, PC-3 cells exhibited a biphasic survival curve atflow rates above 20 μL/sec. Impressively, a very similar shape wasobserved for the survival curves of the other cancer cell lines tested.Meanwhile, cell death in the immortalized, but non-transformed celllines was much more linear. Although the survival curves of primarycells also appear biphasic, nearly 90% of all cells in suspension haddied by the second passage of shear stress, compared to an average ofonly 30% of cancer cells (FIG. 2B). The percentage of cell loss perpassage of shear stress is shown graphically in FIG. 2C.

Resistance to detachment-induced cell death, or anoikis, is a hallmarkof cancer. Therefore, the viability of non-shear stress-treated PC-3cells was compared to the viability of non-transformed cell lines andprimary cells over a one hour period in suspension. The viability of allcells tested was unaffected by detachment within 10 minutes insuspension and there was only a small amount of cell death during a 30minute period, which is the time that it takes to perform the shearstress protocol at the lowest flow rate, which represents the longestassay flow rate (FIG. 2D). Therefore, differences in shear stresssurvival between these cell types are not likely due to exacerbations indetachment-induced cell death.

Whether the biphasic survival response of cancer cells was dependentupon changes which occur in freshly suspended cells was alsoinvestigated. A suspension of PC-3 cells was made and divided it intothree aliquots; shear treatment was started as usual using the firstaliquot. The next was sheared following the initial round, which tookroughly 10 minutes. The third aliquot was held for one hour beforeshearing. Viability data for each aliquot was similar (FIG. 9).

Finally, it is known that serial passage of human or mouse cancer celllines through mice via systemic injection often selects for cells thatexhibit enhanced metastatic potential [Fidler, I. J., Biologicalbehavior of malignant melanoma cells correlated to their survival invivo. Cancer Res, 1975. 35(1): p. 218-24., Fidler, I. J. and M. L.Kripke, Metastasis results from preexisting variant cells within amalignant tumor. Science, 1977. 197(4306): p. 893-5.]. Therefore, thepossibility that experimental metastasis selects for cells of increasedshear stress resistance was tested by comparing the survival of PC-3,MDA.MB.231, and B16.f0 cells with their in vivo derivatives in the shearstress protocol. No appreciable differences in shear stress resistancebetween parent cancer cells versus metastatic derivatives (showedalongside many other cell lines in FIGS. 10A-B) was detected. Thisfinding suggests that metastasis does not select for shear stressresistance, however we note that PC-3 and MDA.231 are both derived frommetastatic tumors, thus these cells have previously experiencedcirculation prior to passage in mice.

Shear Stress Resistance in Carcinoma Cells is Transient and Inducible

The observation that cancer cell death was precipitous over the firsttwo passages of the shear stress protocol, but diminished withsubsequent passage led to the hypothesis that passage selects for shearstress-resistant subpopulations within the cancer cell lines. If thisresistance phenotype has a genetic basis, then cells collected followingthe shear protocol should exhibit enhanced resistance to shear stress.However, this was not found to be the case. In several cell lines,exposing shear stress-surviving cells to a second round of shearprotocol after recovery in culture demonstrated that survival wassimilar to the initial round of the shear protocol (FIG. 3). This datasupports the conclusion that the dramatic reduction in the rate of celldeath observed in cancer cell lines results from a transient,physiologic protective response to shear stress.

Repeated Exposure to Shear Stress Results in Changes in CellularMembrane Integrity and Induced Resistance to Shear Forces

The observation of a biphasic viability curve in cells sheared atgreater than 20 μl/sec led to the hypothesis that cancer cells exposedto shear forces can elaborate changes conferring greater resistance tofuture shear forces, and thereby resist death by avoiding irreparablemembrane damage. Implicit in the hypothesis was the ability of cells torepair a certain degree of membrane damage. To address this hypothesis,the shear protocol was conducted in the presence of propidium iodide(PI). Typically used to mark non-viable cells by virtue of itsmembrane-impermeability, the accumulation of PI within viable cells wasevaluated to represent a cell population that sustained and repairedmembrane damage secondary to shear forces. Before the first, and aftereach subsequent passage, an aliquot of cell suspension was removed forFACS analysis. When analyzing cell suspensions for PI positive cellswithin a gate consistent with viability by forward and side scatterparameters, viable non-sheared PC-3 cells demonstrated minimal PIaccumulation (mean 0.65% of viable cells, n=9, FIG. 4A, upper left).After an initial shear passage, PI accumulated in 7.28% of the viablecell population (FIG. 4A, upper right). With continuing passages, morePI accumulated, ultimately maximized after passage 10 with 36.73% ofviable cells displaying positive staining (FIG. 4A, lower left). Thisdata indicates that previously undamaged cells sustain membrane damagesufficient to allow PI uptake over repeated passages.

To confirm cell viability as gated by forward and side scatter gates,the vital stain, Calcein AM, was employed. Both prior to shearing andafter 10 passages, nearly all of the cells in the gate of interest wereCalcein AM positive (99.8% and 99.2%, respectively), confirming that allof the cells in gates P1+P2 were, indeed, viable (FIG. 4B).

When PI was withheld from cell suspension until after passage 9, lessuptake of the dye into sheared cells was observed than was observedafter passage 1 in constant presence of PI (4.22% versus 7.28%,respectively; FIG. 4A, lower right vs. FIG. 4A, upper right). Whenplotted over repeated passages in constant presence of PI, there wasless PI accumulation per passage, on average, in the intervals frompassage 3-6 and 7-10 (3.25% and 2.92%, respectively) than in the firstpassage alone (6.64%). The gradual diminution of additional PIaccumulation suggests that fewer cells were being damaged sufficientlyto allow PI uptake, reflecting a smaller population eligible formembrane-damaging destruction. If this assumption is true, introductionof PI to cell suspensions at points intermediate in the assay would beexpected to reveal less PI accumulation than observed when PI is addedprior to passage one. In FIG. 4C, PI was added either prior to passage1, 6, 8, or 10, and then sheared to completion (10 passages). Regardlessof when PI was added, the first passage in its presence elicited apronounced increase in PI positivity; however, this increase wassignificantly less than that seen at passage 1 in constant presence ofPI (7.28% +/−1.38% versus 3.62% p6, 4.90% p8, and 4.22% p10). Becausethe FACS analysis was performed on an equal number of viable cells ateach passage, cell death was not responsible for the diminution of PIaccumulation. Rather, these data suggest that during flow, PC-3 cells inthe model experience a range of shear forces; some sufficient to alterthe membrane integrity of a cell, but insufficient to cause irreparablemembrane damage. The reduced uptake of PI into cells over later passagessuggests that PC-3 cells can evoke an induced response that results inraising the threshold for both reparable membrane damage as well asirreversible, lethal damage.

Induced Shear Stress Resistance Requires Extracellular Calcium

Mechanical damage to plasma membranes can be repaired, in certain celltypes, by a fusion of vesicles in a mechanism that requires the influxof extracellular calcium [Bement, W. M., et al., Rehabilitation and thesingle cell. Curr Opin Cell Biol, 2007. 19(1): p. 95-100.]. This healingprocess has been reported to require the activity of Rho-GTPases andactin polymerization at the site of the wound [Terasaki, M., K. Miyake,and P. L. McNeil, Large plasma membrane disruptions are rapidly resealedby Ca2+-dependent vesicle-vesicle fusion events. J Cell Biol, 1997.139(1): p. 63-74.]. Therefore, whether the observed shear stresssurvival response in cancer cells also requires extracellular calciumwas investigated. When PC-3 cells were suspended in nominallycalcium-free PBS and subjected to the shear stress protocol, a moresteady loss of cell viability and an 8-fold increase in total cell deathwas observed (FIG. 5A). When suspensions of cells in PBS aresupplemented with calcium at the same concentration as complete tissueculture medium a survival curve not significantly different from themedia control suspensions was observed (FIG. 5A). These data suggestthat shear stress damage to cancer cells triggers a survival responsewhich requires extracellular calcium.

In complete medium, PC-3 cells exhibit little cell death at the 20μL/sec flow rate (as seen in FIGS. 1A-D). However, when subjected tothis flow rate in calcium-free PBS, these cells exhibit a linear loss ofviability with roughly 35% more cell death than in the presence ofcalcium (FIG. 5B). This finding indicates that the protective shearstress survival response can be triggered at lower magnitudes of shearforce, which may be more commonly encountered physiologically by CTCs.Finally, to test the specific requirement of calcium in thisshear-induced survival response, it was shown that cells suspended inPBS supplemented with barium were equally susceptible to shear stress ascells in nominally calcium-free PBS (FIG. 11).

Induced Shear Stress Resistance Requires Actin Polymerization

Next, whether rearrangements of the cytoskeleton occur in response toshear stress and thereby imparts a protective advantage to cells wasinvestigated. In support of an active biological process, it was foundthat cell suspensions which were held on ice, and thus expected to havereduced signaling kinetics, for 20 minutes prior to the shear protocolexhibited significantly greater loss of cell viability than under usualconditions (data not shown). To directly test the importance ofcytoskeletal remodeling in the hypothesized shear stress response, webriefly treated PC-3 and MDA.MB.231 cells were fully tested withcytochalasin-D prior to the shear stress protocol. During this time,cell viability was not affected by cytochalasin-D treatment. In bothcell lines there was over 3-fold more cell death and an attenuatedbiphasic survival response in cytochalasin-D treated cells versus DMSOtreated controls (FIGS. 6A-B). Thus, shear stress-induced actinpolymerization appears to play a protective role in cancer cells duringconditions of flow.

Summary and Discussion

The result presented above reveal that most cancer cells, regardless oftissue origin and metastatic potential are constitutively resistant toflow conditions with wall shear forces up to ˜500 dyn/cm². At increasingshear forces, up to ˜6,500 dyn/cm², most of these cancer cells exhibit abiphasic loss of viability. This range of shear forces would beconsidered high to very high/supraphysiologic [Kamm, R. D., Cellularfluid mechanics. Annu Rev Fluid Mech, 2002. 34: p. 211-32., Schirmer, C.M. and A. M. Malek, Estimation of wall shear stress dynamic fluctuationsin intracranial atherosclerotic lesions using computational fluiddynamics. Neurosurgery, 2008. 63(2): p. 326-34; discussion 334-5.].Despite the high magnitude of these values, it is important to note thatfreshly prepared red and white blood cells endured these forces greaterthan all cell types tested. During the first two passages of shear flow(each passage roughly between 1 to 6 milliseconds), the rate of celldeath is six times greater than in the subsequent eight passages. After10 passages at the highest shear rate, the average cell death was ˜60%of the total suspension. The shape of these survival curves suggestseither a selective purification of inherently shear stress-resistantcells, or an adaptive resistance to shear stress. To test thesepossibilities, the surviving fraction of cells subjected to 10 passagesof shear stress were sub-cultured and re-exposed to a second round ofshear protocol. In several cancer cell lines it was shown that thisapproach did not enrich for shear stress-resistant cells. This findingled to the conclusion that the basis for the observed biphasic survivalis an inducible and transient response rather than a stable genetictrait.

After shearing cancer cells in the presence of the membrane-impermeabledye, propidium iodide (PI), it was shown that viable cells allow rapidPI uptake during the first passage of shear stress, indicating alteredmembrane integrity. The rate of PI-uptake by viable cells diminishesover repeated passages, suggesting that membrane integrity was induciblyrepaired and maintained after the initial “priming” round of shearstress. In support of this conclusion, cells which had been shearedseveral times prior to the addition of PI allowed much less of this dyein than those cells in the presence of PI throughout the assay. To gainmechanistic insight into this shear-induced resistance to continuedshear stress, cells were suspended in divalent cation-free PBS. Thesecells were roughly 10-fold more susceptible to shear stress and had lostthe biphasic survival response. When supplementing PBS suspensions withcalcium or barium, it was shown that calcium addition selectivelyrestores biphasic shear stress survival.

Whether actin polymerization in response to fluid shear stress plays arole in the inducible shear stress-resistance response was studied.Cells treated briefly with a non-cytotoxic dose of cytochalasin-D weresignificantly more susceptible to shear stress-induced death. Mechanicaldamage to plasma membranes has been shown to induce a repair response incardiomyocytes, skeletal muscle cells, and oocytes [Terasaki, M., K.Miyake, and P. L. McNeil, Large plasma membrane disruptions are rapidlyresealed by Ca2+-dependent vesicle-vesicle fusion events. J Cell Biol,1997. 139(1): p. 63-74.]. In these cell types, extracellular calciumrapidly enters the membrane wound, triggering fusion of intercellularvesicles with the damaged plasma membrane domain. This membrane“patching” mechanism has been shown to require activation of smallGTPases and rearrangements of the actinomysin cytoskeleton [Bement, W.M., et al., Rehabilitation and the single cell. Curr Opin Cell Biol,2007. 19(1): p. 95-100.]. It is possible that cancer cells have adaptedsuch a mechanism to overcome fluid shear stress.

Importantly, it was shown that inducible shear stress resistance isunique to transformed epithelial cells. Non-transformed cell lines andprimary cells of the human breast and prostate were susceptible tomagnitudes of shear force much lower than those required to inducecancer cell death. These cell types exhibited dramatically greater cellloss over the first two passages of shear flow (upwards of 90% inprimary cells) and did not elaborate resistance to shear stress at laterpassages as seen in most carcinoma cell lines. Thus, when comparing thebehavior and survival of normal epithelial cells to carcinoma cells, itcan be concluded that carcinoma cells 1) have an intrinsically higherresistance to shear stress-induced cellular damage, and 2) are capableof responding to damage from shear stress, resulting in a transient butefficient repair mechanism.

Perhaps the most revealing finding of this study was that multiplecancer cell lines, derived from various tissues, and with a wide rangeof metastatic potential, exhibit a similar phenotype of shear stressresistance. One explanation is that the conservation of this phenotypeis intimately linked to cellular transformation. Common transformingoncogenes, such as Ras, AKT, etc. result in constitutive upregulation ofRho-GTPases and changes in cytoskeletal dynamics, and thus cellmorphology and tensegrity [Tzima, E., Role of small GTPases inendothelial cytoskeletal dynamics and the shear stress response. CircRes, 2006. 98(2): p. 176-85., Cain, R. J. and A. J. Ridley,Phosphoinositide 3-kinases in cell migration. Biology of the cell/underthe auspices of the European Cell Biology Organization, 2009. 101(1): p.13-29.]. Mutations which drive primary tumor growth and invasion havebeen shown to co-opt for metastatic behavior, such as extravasation andangiogenesis [Gupta, G. P., et al., Mediators of vascular remodellingco-opted for sequential steps in lung metastasis. Nature, 2007.446(7137): p. 765-70.]. The data suggest that killing of circulatingtumor cells by hemodynamic shear forces is much lower than oftenestimated. This would argue that survival of such forces is not a largedeterminant of metastatic inefficiency.

The topic of metastatic inefficiency is clinically relevant to the studyof circulating tumor cells (CTCs). Recently, there has been considerableinterest in isolating and quantifying CTCs to develop new prognostic andpredictive tools. One of the largest challenges here is that the merepresence of CTCs in the blood of patients does not always correlate withpoor prognosis or metastasis. As an example, the number of circulatingcells prior to surgery was shown to be predictive of relapse-freesurvival in breast cancer patients [Cristofanilli, M., et al.,Circulating tumor cells, disease progression, and survival in metastaticbreast cancer. N Engl J Med, 2004. 351(8): p. 781-91.]; and surgeries toremove non-small cell lung carcinoma have been reported to causeincreased numbers CTCs in patients, which correlates with relapse[Rolle, A., et al., Increase in number of circulating disseminatedepithelial cells after surgery for non-small cell lung cancer monitoredby MAINTRAC(R) is a predictor for relapse: A preliminary report. World JSurg Oncol, 2005. 3(1): p. 18.]. Conversely, patients with ovarian orcolon cancer who have surgical venous shunts, which introduce many(estimated up to millions) cancer cells into the blood every day, rarelydevelop metastatic disease [Tarin, D., et al., Clinicopathologicalobservations on metastasis in man studied in patients treated withperitoneovenous shunts. Br Med J (Clin Res Ed), 1984. 288(6419): p.749-51., Tarin, D., et al., Mechanisms of human tumor metastasis studiedin patients with peritoneovenous shunts. Cancer Res, 1984. 44(8): p.3584-92.]. These data call into question the fate of CTCs which do notcomplete all steps of metastasis. At the simplest level, it is likelythat these cells are either eventually killed in the bloodstream or thatonce colonizing a secondary tissue that they are unable to proliferatesufficiently to develop into a metastases.

By extending these finding to a clinical context, it is possible thatall CTCs endure shear stress quite well, regardless of their metastaticcapabilities. Thus when analyzing a patient's blood for the presence ofCTCs it should be considered that the absolute number of cells detectedis a much less meaningful prognostic readout than understanding theability of these circulating cells to extravasate and proliferate at adistant tissue. Furthermore, the observation that transformed cancercell lines are considerably more resistant to shear stress than primarycells, supports the conclusion that this biology could be exploited topurify CTC preparations of non-transformed cells, improving theprognostic value of this assay.

Example 2

This example expands on the experiments described in Example 1 withadditional tests of the loss in cell viability due to fluid shear stressfor primary epithelial cells relative to cancerous epithelial cells.

FIGS. 13A-B show the results of cell viability measurements for primarycells of the human breast and prostate (HMEC and PrEC, respectively)after repeated passages through the shear stress protocol at a flow rateof 20 μL/sec. Suspensions of the HMEC and PrEC cells were subjected toten passages and viability was measured at every other passage. For thedata shown, p<0.05 vs. PC-3 (one-way ANOVA, Bonferroni's post tests).For each cell line, n=4 experiments using the syringe pump protocol. Allerror bars=±SEM. As shown in FIG. 13A, at the 20 μL/sec flow rate,primary epithelial cells exhibited a pronounced, but biphasic loss ofviability. In contrast, little loss in viability was observed for thePC-3 cells experiencing the same shear stress protocol, as shown in FIG.13B.

Next, the ability of the shear stress protocol to selectively killnon-cancerous cells in a fluid preparation was demonstrated by carryingout the protocol on a mixture of differentially labeled PrEC and PC-3cells. Viability analysis of mixed cell populations: PC-3 and PrEC cellswere labeled with calcein AM (Invitrogen #C34852) and cell trackerorange (Invitrogen #C2927), respectively. Suspensions, prepared asdescribed in Example 1, were mixed ˜1:1 prior to subjecting to the FSSprotocol. To assess cell viability using flow cytometry, viable, calceinAM⁺ (green) cells and viable, cell tracker orange⁺ (orange) cells werecounted. The number of green or red cells was divided by the totalnumber (green +orange) stained viable cells counted at each passage todetermine the relative numbers of each cell type in the mixture. Before(P.0) and after ten passages (P.10) of FSS, 10,000 fluorescent eventswere counted using flow cytometry. The results are shown in FIG. 14A andFIG. 14B.

After exposure to FSS the ratio of PC-3 (FIG. 14A, bottom rightquadrant) to PrEC (FIG. 14A, top left quadrant) changed from 0.955 to2.80. Averaged results of three independent experiments show a change inthis ratio from 1±0.07 to 3.13±SEM=0.4. See also FIG. 14B, 25 μL ofmixed (PC-3:PrEC) cell suspension was plated into collagen 1-coated8-well chamber slides before (P.0) and after ten passages (P.10) of FSS.These cells were allowed to adhere overnight and were then fixed in 4%paraformaldehyde for 10 minutes. Fixed cells were counterstained withDAPI and imaged using the Cy2 filter on a Leica DME 2500. For threeseparate experiments, 5 fields of view were imaged for P.0 and P.10suspensions. At P.0 the ratio of calcein AM positive to negative cellswas very close to 1. All error bars=±SEM.

Example 3

This example expands on the experiments described in Example 1 withadditional tests showing that fluid shear stress resistance can becorrelated to transforming oncogene, including ras, myc and P13K. Thiswas shown using prostate and melanoma cells specifically engineered toexpress transforming oncogenes.

LH,LHSR and LHMK cells were obtained from Dr. William Hahn (Dana FarberCancer Institute) and R545 cells were obtained from Dr. Lynda Chin (DanaFarber Cancer Institute) and cultured as described in Berger R, et al.(2004) Androgen-induced differentiation and tumorigenicity of humanprostate epithelial cells. Cancer Res 64(24):8867-8875 and Chin L, etal. (1999) Essential role for oncogenic Ras in tumour maintenance.Nature 400(6743):468-472. The fluid shear stress protocol and cellviability analysis were carried out as described in Example 1.

The effect of FSS (at 250 μL/sec) was compared between wild type primaryhuman prostate epithelial cells (PrEC), immortalized PrEC (LH), andMyc/PI3K (LHMK) or Ras (LHSR) transformed PrEC. ***,p<0.001 vs. WT; #,p<0.05; ###, p<0.001 vs. LH;

, p<0.05 vs. LHSR (One way ANOVA, Bonferroni post tests). R545 melanomacells (derived from Tyr/Tet-Ras INK4a−/−mice) express H-Rasv12 in adoxycycline-dependent manner. These cells were cultured for two passagesin the presence or absence of 2 μg/mL doxycycline before shearing at 250μL/sec. n=4 for all cell lines and conditions using syringe pump.

As shown in FIG. 15A, immortalization of these cells via expression ofSV40 large T antigen and hTERT did not significantly affect FSSresistance, but transformation via MYC/PI3K or H-ras led to robustresistance to FSS similar to that seen in cancer cell lines. Similarresult in a mouse melanoma cell line in which H-RasV12G expression isunder the control of a tetracycline-inducible promoter. In the presenceof doxycycline, H-Ras-expressing cells exhibited increased resistance toFSS, as shown in FIG. 15B.

The involvement of the actin cytoskeleton in FSS resistance provides anavenue to explain the role of transforming oncogenes in this process.Ras and PI3K are well known to influence cytoskeletal dynamics.Interestingly, various biophysical measurements indicate thattransformed cells are more deformable (less stiff) than theirnon-tumorigenic counterparts. This is commonly interpreted as favoringan invasive and migratory phenotype, but such deformability may lead tosusceptibility to high FSS damage, disfavoring hematogenousdissemination. Oncogenic activation may result in cells poised torapidly mobilize the actin cytoskeleton in response to calcium influxdue to high FSS.

Example 4

This example expands on the experiments described in Example 1 withadditional tests showing that the loss of cell viability observed afterthe fluid shear stress was not a function of pH or temperature.

Unlike the cancerous cells studied in the previous examples, which werein media buffered with sodium bicarbonate, PC-3 cells in this examplewere suspended in DMEM/F12, 10% FBS in the presence or absence of 20 mMHEPES (avg. pH at room temperature: 7.3 vs. 7.7, respectively). As shownin FIG. 16A, no significant difference in cell survival was observedunder these two conditions. In addition, PC-3 cells suspended in regularmedia were also sheared at 37° C. in a walk-in incubator. As shown inFIG. 16A, a biphasic FSS response was still observed. *, p<0.05 vs.DMEM/F12 room temp, n=4 using the pump method (repeated measures ANOVA,Bonferroni's post tests). For comparison, primary cells (HMEC, n=2 andPrEC, n=5 experiments using pump method) were subjected to the sameshear stress protocol at 37° C. and room temperature. As shown in FIG.16B, cell survival for the primary cells was significantly reduced atboth temperatures compared to their cancerous counterparts.

Example 5

This example illustrates the use of the present methods to purify apreparation comprising a fluid suspension of tumor cells isolated from asolid tumor.

Epithelial Tumor Cells Freshly Isolated from Murine Prostates DisplayBiphasic Loss of Viability when Exposed to Fluid Shear Stress.

Aged mice harboring Pten-deficient prostates were sacrificed (details onmice as previously published in Svensson, R. U., Am. J. Pathol. 2011).Prostates were removed and epithelial cells isolated using a mechanicaland chemical (collagenase IA) approach (see, Lukacs, R. U. et al., Nat.Protoc. 2010). A single cell suspension was exposed to fluid shearstress (FSS) at a flow rate of 250 μl/sec according to the methodspresented in Example 1. Viability was assessed by bioluminescenceimaging by virtue of a prostate epithelium-specific expression ofluciferase. As previously demonstrated in transformed cultured cells,these transformed cells exhibit the biphasic survival curve indicativeof an early FSS-induced elaboration of resistance to subsequentexposures to FSS. This data, which is shown in FIG. 17, supports theconclusion that inducible resistance is conferred by transformation,rather than an artifact of in vitro culture. Furthermore, itdemonstrates that the present methods can be used for quicklydistinguishing benign from malignant cells from clinical biopsies.

The word “illustrative” is used herein to mean serving as an example,instance, or illustration. Any aspect or design described herein as“illustrative” is not necessarily to be construed as preferred oradvantageous over other aspects or designs. Further, for the purposes ofthis disclosure and unless otherwise specified, “a” or “an” means “oneor more”. Still further, the use of “and” or “or” is intended to include“and/or” unless specifically indicated otherwise.

The foregoing description of illustrative embodiments of the inventionhas been presented for purposes of illustration and of description. Itis not intended to be exhaustive or to limit the invention to theprecise form disclosed, and modifications and variations are possible inlight of the above teachings or may be acquired from practice of theinvention. The embodiments were chosen and described in order to explainthe principles of the invention and as practical applications of theinvention to enable one skilled in the art to utilize the invention invarious embodiments and with various modifications as suited to theparticular use contemplated. It is intended that the scope of theinvention be defined by the claims appended hereto and their equivalents

What is claimed is:
 1. A method for purifying a fluid sample comprisingextracellular calcium and cells of a mammalian subject, the cellscomprising viable normal cells, the method comprising: applying aninitial pulse of fluid shear stress to the sample in vitro, wherein theinitial pulse of fluid shear stress is sufficient to induce a fluidshear stress resistance in viable cancerous cells present in the samplesuch that the magnitude of shear stress required to kill the viablecancerous cells is greater than the magnitude of shear stress requiredto kill the viable normal cells; and subsequently applying one or moreadditional pulses of fluid shear stress to the sample in vitro, saidfluid shear stress sufficient to selectively kill the viable normalcells relative to the viable cancerous cells such that the ratio of theviable cancerous cells to the viable normal cells is increased.
 2. Themethod of claim 1, wherein the cells of the mammalian subject compriselung cells.
 3. The method of claim 2, wherein the mammalian subject is ahuman.
 4. The method of claim 1, wherein the fluid sample is an in vitrofluid preparation.
 5. The method of claim 4, wherein the in vitro fluidpreparation is prepared from a solid tissue biopsy.
 6. The method ofclaim 1, wherein the cells of the mammalian subject comprise the viablecancerous cells in addition to the viable normal cells.
 7. The method ofclaim 1, wherein the viable cancerous cells are viable cancerousepithelial cells.
 8. The method of claim 1, wherein the ratio of theviable cancerous cells to the viable normal cells in the sample is atleast doubled.
 9. The method of claim 1, wherein the percent viabilityfor the viable cancerous cells is at least 40% and the percent viabilityfor the viable normal cells is no greater than 20% substantiallyimmediately following the application of the initial and additionalpulses of fluid shear stress, wherein percent viability is determined asthe percent viability relative to a control maintained without theapplication of shear stresses for the duration of the pulseapplications.
 10. The method of claim 1, wherein the initial andadditional pulses of fluid shear stress apply a fluid shear stress inthe range from about 500 dyn/cm² to about 6500dyn/cm².
 11. The method ofclaim 1, wherein the additional pulses apply a supra-physiologic levelof fluid shear stress.
 12. The method of claim 1, wherein the steps ofapplying the initial pulse of fluid shear stress to the sample andapplying one or more additional pulses of fluid shear stress to thesample comprise passing the sample through a conduit at a substantiallyconstant flow rate.
 13. The method of claim 1, wherein the initial pulseand the one or more additional pulses each have a duration of no greaterthan about 20 msec.
 14. The method of claim 1, wherein at least twoadditional pulses of fluid shear stress are applied to the sample. 15.The method of claim 1, further comprising subsequently measuring theconcentration of the viable cancerous cells in the sample.
 16. Themethod of claim 1, further comprising subsequently conducting an assayon the sample, the assay providing a cancer diagnosis or a cancerprognosis for the mammalian subject.